The transcription factor Pax5 is a master regulator of vertebrate B cell development and activation, targeting a large number of genes. Inappropriate expression of Pax5 has been correlated with B-lymphoid malignancies. The human Pax5 gene undergoes extensive alternative RNA splicing, resulting in Pax5 proteins that differ in their trans-activation potential. B cell lymphomas and multiple myelomas have altered expression of alternative Pax5 splice forms but correlations between individual splice forms and disease are unclear. Our laboratory studies have focused on B cell development and activation using a well-defined, alternative animal model, the rainbow trout. Preliminary data show that B cells from rainbow trout express alternative Pax5 isoforms identical or similar to those detected in humans, but not observed in mice. Almost all human and trout Pax5 splice forms span either the trans-activation and/or repressor domain of Pax5. These domains regulate the activity of Pax5 and consequently, must modulate expression of Pax5 target genes. During the past grant period, an approach was developed to identify the main differentiation stages of individual trout B cells, using flow cytometric analysis. In the absence of available cell surface markers, a combination of stage-specific, highly conserved intracellular markers was used to identify each differentiation stage. Markers included IgM, Pax5, XbpI, RAG1, and BrdU. With that approach now in place, the following three research aims will be addressed: 1). The isolation of additional Pax5 isoforms, and determination of their transcript levels in trout immune tissues. 2). Determination of the transactivation potential of trout Pax5 isoforms using transient transfections. 3). Development of isoform-specific anti-Pax5 antibodies to determine when each B cell expresses a particular Pax5 isoform. Flowcytometric analyses on the patterns of Pax5 isoform expression will be studied in trout infected with a bacterial pathogen. The strength of this approach is based on the ability to analyze expression of Pax5 isoforms in individual cells. The proposed studies will for the first time link Pax5 isoform expression with the developmental and/or activation state of individual B cells. Relevance of the studies includes increased knowledge on the functional significance of alternative Pax5 splicing in B lineage cells in infectious disease and malignancy, as well as development of diagnostic antibody tools, both in humans and in fish. PUBLIC HEALTH RELEVANCE: The transcription factor Pax5 is expressed in the antibody-producing (B) cells of vertebrates, and is alternatively spliced. In humans, abnormal splice forms of Pax5 have been detected in B cell lymphomas and multiple myelomas. Studying Pax5 isoforms in fish, which use similar isoforms as humans, helps us to understand their function both during cell development and during malignancy.